Neointimal hyperplasia is a significant problem that results in the failure of vascular interventions. The classic arterial injury response that leads to the development of neointimal hyperplasia describes processes involving the intima and media with a relative lack of involvement of the adventitia. However, the adventitia is no longer considered a simple structural component of the arterial wall. In the last decade, research has shown that many cell types from the adventitia actively regulate and contribute to the development of neointimal hyperplasia, including resident and circulating adventitial stem and progenitor cells. We, and others, have demonstrated that NO is a potent inhibitor of neointimal hyperplasia through regulating different aspects of the classic arterial injury response. However, little is known about how NO affects the adventitial layer of the arterial wall following injury. We have recently assessed the cellular response throughout the arterial wall to injury and exposure to NO. Interestingly, while NO prevents the development of neointimal hyperplasia and delays repopulation of the media, we were surprised to find that NO actually increases cellularity in the adventitia. This increase in cellularity is not due to an increase in vascular smooth muscle cells, fibroblasts, myofibroblasts, or inflammatory cells, but may in fact be due to an increase in Sca1+ progenitor cells, among others. Thus, given the important role of the adventitia in regulating the arterial injury response, recent discoveries of the role of stem and progenitor cells in this process, and our preliminary data, we hypothesize that NO inhibits neointimal hyperplasia by regulating recruitment and phenotypic differentiation of resident and circulating adventitial stem and progenitor cells following injury. Furthermore, we hypothesize that NO supports differentiation of adventitial stem and progenitor cells into endothelial-like cells, contributing to adventitial neovascularity. To investigate these hypotheses, our specific aims are: 1) to characterize the effect of NO on Sca1+, CD34+, and flk-1+ adventitial stem and progenitor cell populations following arterial injury in vivo; 2) to determine the effect of NO on Sca1+, CD34+, and flk-1+ adventitial cell populations in vitro; and 3) to manipulate Sca1+, CD34+, and flk-1+ adventitial cell populations in vivo to determine if NO-mediated inhibition of neointimal hyperplasia following arterial injury is dependent on these adventitial cell populations. The innovative studies described in this proposal will result in a change in how we think about the adventitia and the vascular biology of NO, and provide a novel mechanism by which NO regulates neointimal hyperplasia that will lead to the development of new strategies to prevent neointimal hyperplasia and restenosis following vascular interventions. PUBLIC HEALTH RELEVANCE: Recent studies have uncovered the importance of the adventitia as well as resident and circulating stem and progenitor cells in mediating the arterial injury response that leads to the development of neointimal hyperplasia. Nitric oxide (NO) is a potent inhibitor of neointimal hyperplasia, but little is known about how NO affects the adventitial layer of the arterial wall following injury. The studies in this proposal will elucidate the role of adventitial stem and progenitor cells in mediating the beneficial effects of NO in the vasculature and will result in a change in how we think about the adventitia and the vascular biology of NO thereby allowing the development of new strategies to prevent neointimal hyperplasia and restenosis following vascular interventions.